CRISPR cas, a potential tool for targeted genome modification in crops.

CRISPR –casA Potential Tool for Genome Modification

MONOJ SUTRADHAR

PALB 3243

Sr. M.sc, Plant Biotechnology

UAS, GKVK,Bangalore

Department of Plant Biotechnology,UAS,GKVK,Bangalore25 October 2014

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Contents

Introduction

History

Mechanism overview

Types of CRISPR-cas system

Cas9 nuclease

Comparisons among different kinds of nucleases

Case study

Conclusion

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Department of Plant Biotechnology,UAS,GKVK,Bangalore2

CRISPR

• It represents a family of DNA repeats in most archaeal (~90%) and bacterial (~40%) genomes provides acquired immunity against viruses and phages.

(Barrangou et al.,2010)

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CRISPRs (clustered regularly interspaced short palindromic repeats) are DNA loci

containing short repetitions of base sequences which separated by short "spacer

DNA" from previous exposures to a virus or phage.

25 October 2014

Department of Plant Biotechnology,UAS,GKVK,Bangalore

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The size of CRISPR repeats and spacers varies between 23 to 47

base pairs (bp) and 21 to 72 bp, respectively. Generally, CRISPR

repeat sequences are highly conserved within a given CRISPR

locus.

• The CRISPR leader, defined as a low-complexity, A/T-rich, noncoding sequence, located immediately upstream of the first repeat, likely acts as a promoter for the transcription of the repeat-spacer array into a CRISPR transcript, the pre-crRNA.

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The full-length pre-crRNA is subsequently processed into specific small RNA molecules that correspond to a spacer flanked by two partial repeats.

HISTORY

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Zhang et al.,2014

Types of CRISPR CAS system

There are three types of CRISPR-Cas systems, which vary in their specific target and mechanism of action.

Type I systems cleave and degrade DNA,

Type II systems cleave DNA ,

Type III systems cleave DNA or RNA .

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Type I and II systems require two principal factors to effectively target DNA:

(i) complementarity between the CRISPR RNA spacer and the target “protospacer” sequence.

(ii) a protospacer-adjacent motif (PAM) specific to each CRISPR-Cas system flanking the protospacer.

•Effective targeting can occur even for multiple mismatchesbetween the CRISPR RNA and the protospacer, althoughmismatches within the “seed” region flanking the PAM aremore disruptive .


Similar factors are required for DNA-targeting by type III systems, where these systems evaluate base pairing between the target sequence and the region flanking the protospacer.


Mechanistically, although defense is spacer-encoded, the information that lies within the CRISPR repeat- spacer array becomes available to the Cas machinery through transcription

Mechanism of CRISPR-cas system


CRISPR-Cas systems are RNA-directed adaptive immune systems in many bacteria and most archaea that recognize nucleic acids of invading plasmids and viruses.


Recognition is directed by CRISPR RNAs that are processed from transcribed arrays of alternating target specific“spacer”sequences and identical “repeat” sequences.


The spacer region of each CRISPR RNA base pairs with complementary nucleic acids, driving cleavage or degradation by the Cas proteins within minutes of invasion.


Barrangou et al.,2012

DNA repair


Zhang et al.,2014


CRISPR interference


RNA programmable gene knockdown by Type iii-B Cmr CRISPR-cas system.

Terns et al.,2014


The Cmr effector complex(blue) comprises six Cmr proteins and a crRNA.

The guide region of crRNA base-pairs with the homologous sequence in the mRNA target and the target RNA is cleaved by the complex.

Applications of Cmr system include RNA-directed gene knockdown to investigate gene function or to facilitate metabolllic engineering.

25 October 2014Department of Plant Biotechnology,UAS,GKVK,Bangalore

In bacterial type 2 CRISPR cas system, the site specificity is defined by complementery base pairing of a small CRISPR RNA(crRNA

After annealing to atransactiviting CRISPRRNA(tracrRNA) the crRNA directlyguides the cas9 endonuclease tocleave the targeted DNA sequence.

The crRNA–transcr- RNAheteroduplex could be replaced byone chimeric RNA (so-called guideRNA (gRNA)) and the gRNA couldbe programmed to target specificsites.

Jinek et al.,2012

25 October 2014 Department of Plant Biotechnology,UAS,GKVK,Bangalore 24Joung et al.,2012

Comparision between sgRNA and crRNA-tracrRNA hetero duplex

•Advantages• Flexible targeting

• Sequence specific

• Transferable(codon optimized,NLS)

• Efficient

• Precise cleavage

• Affordable

• Quick

• Multiplex guides

• Multiplex orthogonal system


Caveats Cas9 is a large protein PAM – dependent design limitations Off –target cleavage

The CRISPR/cas9 system has also been used in model plants like Nicotiana benthamiana, Arabidopsis thaliana and crops like wheat,rice,sorghum by transient or stable transformation.


In Humans,Zebrafish,Drossophilla,Mice,rats genome editing has been achived by combining Streptococcus pyogenes cas9 and a synthetic single guide RNA(sgRNA) consisting of CRISPR RNA(crRNA) and tracrRNA.

(Cong et al.,2013),(Jiang et al.,2013),

(Joung et al.,2013),(Gartz et al.,2013), (Susan et al.,2014)

Other nucleases


Sequence-specific nucleases increase the efficiency of gene targeting. Among them, zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) are the two most commonly used sequence-specific chimeric proteins.


Gao et al.,2014

In general, single zinc-finger motif specifically recognizes 3 bp, and engineered zinc-finger with tandem repeats can recognize up to 9–36 bp. However, it is quite tedious and time-consuming to screen and identify a desirable ZFN (Pabo et al., 2001).


Once the ZFN or TALEN constructs are introduced into and expressed in cells, their programmable DNA-binding domains can specifically bind to a corresponding sequence and guide the chimeric nuclease (e.g. FokI nuclease) to make a specific DNA strand cleavage.

CASE STUDY


Introduction

The bacterial Type II cluster regularly interspaced short palindromicrepeats (CRISPR)-associated nuclease (Cas) is emerging as an efficient toolfor genome editing in microbial and animal systems as well as in plants.


Three guide RNAs (gRNAs) with a 20–22-nt seed region were designed to pair with distinct rice genomic sites which are followed by the PAM.


The secondary structure of gRNA mimics the crRNA–transcrRNAheteroduplex that binds to Cas9.

The 5′-end of gRNA (gRNA seed) pair with one strand of targeted DNA. The scaffold of gRNA is labeled with dark-red cycles.A PAM motif (N-G-G) is located adjacent to the DNA– gRNA pairing region in the complementary strand of targeted DNA.

The Cas9 nuclease would cleave both strands of DNA at a conserved position which is 3 bp to the PAM motif.


pRGE vectors for transient expression.

A DNA dependent RNA polymerase III promoter and terminator are used to control the transcription of engineered gRNA.


Rice pol III promoters(snoRNA U3 and U6 promoters) were isolated to make pRGE3 and pRGE6 vectors.

Plant DNA dependent RNA polymerase II(pol II) promoter and terminator are used to control the expression of a chimeric cas9 nuclease

hspCas9 encodes a human codon optimized Cas9 nuclease which includes a nuclear localization signal(NLS) and a FLAG tag. Amp represents an ampicillin resistance gene.

The designed oligonucleotides duplex can be inserted into Bsal sites in pRGEvectors and fused with gRNA scaffold to construct engineered gRNA.


The sequence with red colour will be replaced by designed DNA sequence encoding gRNA.

Italic lower case letter indicates overhang sequence after Bsaldigestion


Design of gRNAs to target three specific sites of OsMPK5.

The targeted sites by engineered gRNA(PS1-3) are shown as PS1,PS2 and PS3.

• PS1 contains a Kpnl site and PS3 contains a Sacl site.


The rectangles represent exons of which the black ones indicate the OsMPK5 coding region.

F-256 and R-611 indicate the position of primers used to amplify genomic fragment of OsMPK5.

• Base pairing between the seed region of engineered gRNAsand the targeted sites in the OsMPK5 gene.


PS1-gRNA was paired with the coding strand of OsMPK5 whereas PS2 and PS3 gRNA were paired with the template strand of OsMPK5.

The predicted gRNA-Cas9 cutting position was indicated with the scissor symbol. The PAM was shown in red colour


Detection of Genome editing and targeted mutations at the PS1 and PS3 sites in the OsMPK5 locus by RE-PCR.

Results

The engineered gRNAs were shown to direct the Cas9 nuclease for precisecleavage at the desired sites and introduce mutation (insertion or deletion)by error-prone non-homologous end joining DNA repairing with 3–8%efficiency.


Further analysis suggests that mismatch position between gRNAseed and target DNA is an important determinant of the gRNA–Cas9 targeting specificity, and specific gRNAs could be designed to target more than 90% of rice genes.

CONCLUSION

The CRISPR cas utilize guide RNAs to effectively recognize and target foreign DNA and RNA for destruction.

RNA guided recognition make this immune system highly and rapidly adaptable to diverse targets(recognizing new targets require guide RNA sequence which can be obtained directly from invader).

Flexible and accessible tool for multiple application like genome editing and modulation of gene expression.

Notably understanding of the multiple CRISPR cas system is far from complete and additional tools and applications are yet to come from fertile research.

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CRISPR cas, a potential tool for targeted genome modification in crops.

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